Methods and compositions for inhibiting cellulollytic symbionts

ABSTRACT

Shipworms are important destroyers of wood in the marine environment. Wood users have long sought methods for preventing or limiting their attack. The invention is directed to compositions and methods for preventing or limiting cellulolytic degradation by inhibiting cellulolytic organisms, in particular symbiont organisms. The invention discloses methods and compositions to inhibit the growth of a shipworm symbiont responsible for wood degradation.

[0001] This application claims priority to application Ser. No.60/282,962, filed Apr. 11, 2001, the disclosure of which is incorporatedherein by reference in its entirety.

[0002] The work to develop the invention was supported through a grantfrom the National Oceanic and Atmospheric Administration. The U.S.government may have certain rights in the invention.

FIELD OF THE INVENTION

[0003] The invention relates to compositions and methods of woodpreservation in terrestrial and aquatic environments. Moreover, theinvention relates to methods for screening compositions that may beuseful for wood preservation, and to compositions identified thereby.

BACKGROUND OF THE INVENTION

[0004] The protection of wood exposed in aquatic and terrestrialenvironments has long posed a challenge. For centuries, marine borerswere a major cause of deterioration of wooden ships and the economicwell being of seafaring nations depended upon their ability to maintaina sea-worthy fleet. While wooden ships no longer play a major role inmaritime commerce, wood forms an important component of the marineinfrastructure, particularly in underdeveloped countries. In thedeveloped countries, wooden ships, pilings, and buildings are importantcomponents of authentic reconstruction of historical towns and fishingvillages. Wood used in these exposures is typically treated with highlevels of preservatives that can include creosote, chromated copperarsenate, or ammoniacal copper zinc arsenate. While these chemicals arevery effective, concerns have arisen about the potential for theirmigration into the surrounding water column and the effect on non-targetmarine organisms. There remains a public demand for the development ofless toxic marine wood treatments.

[0005] Similarly, degradation of wood in terrestrial environments by theaction of termites and other insects has been met by use of lumberpressure treated with many of the above agents, including arsenates.Contamination of soils and fresh water supplies from leaching frompressure treated lumber is an environmental concern.

[0006] One problem with developing alternative treatments is a lack ofknowledge about how the current treatments affect various marine borersand terrestrial insects. This lack of knowledge stems from thewidespread effectiveness of the chemicals currently used for thispurpose, which has up to now suppressed development of commerciallyeconomic, environmentally safer alternatives. Aquatic wood protection,and wood protection in general, has tended to develop by increasing theamount of preservative to achieve a toxic threshold, then furtherincreasing the preservative or treatment to ensure that most of thetreated wood achieves this level of protection. This approach results inlittle concern for the effects of the toxicant on the target organism.Applicants found that the development of alternative control strategiesfor marine environments can be accomplished by a more intimate knowledgeof marine borer biology.

[0007] Teredinid bivalves (shipworms) are important degraders of wood inmarine environments. Unlike other marine wood borers, shipworms use woodas a food source, but they do so via symbiotic cellulolytic nitrogenfixing bacteria. Waterbury, et al. A cellulolytic nitrogen- fixingbacterium cultured from the Gland of De shayes in shipworms(Bivalvia:Tereinidae), 221 Science 1401 (1983). The role of these bacteria inshipworm biology, particularly as it relates to substrate selection,remains poorly understood. Similarly, termites use a flagellatedprotozoan (genus Trichonympha) as symbiont.

[0008] The invention discloses tests to identify inhibitors of asymbiont cellulolytic organism and to identify new compounds for woodtreatment to limit or prevent degradation, preferably compounds thathave fewer environmental problems that those presently used.

SUMMARY OF THE INVENTION

[0009] The invention is directed generally to methods for identifyingcompositions useful for inhibiting wood degradation, and to thecompositions identified by the methods.

[0010] In one aspect, the invention is directed to a method foridentifying a composition for limiting or preventing shipworm damagecomprising the steps of: (a) incubating a shipworm symbiont in theabsence and presence of a test composition under conditions such thatthe shipworn symbiont grows, has metabolic function, or both, in theabsence of the test composition and (b) comparing the growth, metabolicfunction, or both, in shipworm symbionts in the absence and presence ofthe test composition; wherein if the growth, metabolic function, orboth, in shipworm symbionts is less in the presence of the testcomposition than in the absence of the test composition, the testcomposition is a composition for limiting or preventing shipworm damage.In one aspect the conditions are limited to measurement and comparisonof the growth of the shipworm symbiont. In one aspect the viability ofthe symbiont is measured and compared. In a preferred aspect, theinvention is directed to a method wherein the symbiont is aTeredinibacter. In a preferred aspect, the invention is directed to amethod wherein the test composition is a wood extract. In a morepreferred aspect, the invention is directed to a method wherein the woodextract is an aqueous extract from a wood selected from the groupconsisting of Douglas-fir, ebony, jarrah, koa, lignum vitae, narra,Osage orange, paduccah, purpleheart, red alder, red oak, teak, verawood,and western redcedar. In a yet more preferred aspect, the invention isdirected to a method wherein the wood is selected from the groupconsisting of jarra, koa, red oak, and western redcedar. In a still morepreferred embodiment, the wood extracts have been analyzed and specificcompounds within the extract have been identified that function toinhibit or prevent wood degradation.

[0011] In one aspect, the invention is directed to a compositionidentified by a method comprising the steps of: (a) incubating ashipworm symbiont in the absence and presence of a test compositionunder conditions such that the shipworm symbiont grows, has metabolicfunction, or both, in the absence of the test composition and (b)comparing the growth, metabolic function, or both, in shipworm symbiontsin the absence and presence of the test composition; wherein if thegrowth, metabolic function, or both, in shipworm symbionts is less inthe presence of the test composition than in the absence of the testcomposition, the test composition is a composition for limiting orpreventing shipworm damage. In a preferred aspect, the composition doesnot consist essentially of a copper-containing compound, achromium-containing compound, an arsenic-containing compound, creosote,or combinations thereof. In one aspect, the invention is directed to amethod of wood preservation comprising treating the wood with acomposition identified by the above assay method.

[0012] In another aspect, the invention is directed to a method foridentifying a composition for inhibiting growth, metabolic function, orboth, of a cellulolytic organism capable of existing as a symbiontcomprising the steps of: (a) incubating a cellulolytic organism capableof existing as a symbiont in the absence and presence of a testcomposition under conditions such that the cellulolytic organism grows,has metabolic function, or both, in the absence of the test composition;and (b) comparing the growth, metabolic function, or both, in thecellulolytic organism in the absence and presence of the testcomposition; wherein if the growth, metabolic function, or both, in thecellulolytic organism is less in the presence of the test compositionthan in the absence of the test composition, the test composition is acomposition for inhibiting growth, metabolic function, or both, of thecellulolytic organism. The cellulolytic organism capable of existing asa symbiont can be an isolate of a species occurring as a symbiont. Thecellulolytic organism can be a prokaryote or eukaryote. The natural hostorganism can be in the animal kingdom, that is metazoan, including butnot limited to mollusca and arthropoda. In one aspect the cellulolyticorganism is an intracellular symbiont. In another aspect thecellulolytic organism is an extracellular symbiont.

[0013] In a preferred aspect, the invention is directed to a compositionidentified by one or more of the methods of the invention. In a morepreferred aspect, the composition does not consist essentially of acopper-containing compound, a chromium-containing compound, anarsenic-containing compound, creosote, or combinations thereof. In amore preferred aspect, the invention is directed to a method of woodpreservation comprising treating the wood with the composition.

[0014] In one aspect, the invention is directed to a compositioncomprising an aqueous extract of a wood wherein the extract is capableof inhibiting the growth, metabolic function, or both, of a cellulolyticorganism. In one embodiment, the cellulolytic organism is capable ofexisting as a symbiont. In a preferred embodiment, the symbiont is ashipworm symbiont.

DETAILED DESCRIPTION

[0015] In one aspect, the invention is directed to a method foridentifying a composition that can be used to limit or prevent shipwormdamage. The method advantageously uses Teredinibacter, and in oneembodiment uses any species of Teredinibacterisolated from B. setacea.Any cellulolytic organism can be used that is capable of existing as asymbiont with a host organism, however, may be used in the presentinventive methods. Teredinibactermay be grown from a single colony andcan be selected for desirable traits. In the alternative, a mixedpopulation of Teredinibactermay be used for the assay. Moreover, otherspecies or a combination of species are suitable as long as they are ina symbolic relationship with a wood-degrading or wood damaging hostorganism.

[0016] Variants of the symbiont, including variants selected forexpression or lack of expression of genetic traits, are suitable for usein the invention. Genetically engineered variants of the symbiont arealso suitable. Such variants can be obtained by standard methods.

[0017] The incubation of the symbiont can be at, below, or aboveambient, including, but not limited to from about 4° C. to about 40° C.In one embodiment the incubation temperature is between about 16° C. andabout 32° C. In one embodiment, the incubation temperature is betweenabout 24° C. and about 28° C. The incubation can be performed in liquidsuspension, in semi-solid suspension, or on plates. In one aspect, theincubation of the symbiont is carried out on agar plates. Thedetermination of inhibition can be by any method known in the art,including detecting a zone of inhibition, a zone of effect, plaqueformation, plaque inhibition, growth, inhibition of growth, catabolismof substrates, generation of metabolic products, and inhibition ofgeneration of metabolic products. The inhibition can be total orpartial. In one embodiment the incubation is at 26° C. for two daysusing a lawn of symbiont and detecting inhibition by bacterial lysis.

[0018] The Teredinibacter, or other symbiont, may be propagated by meansstandard in the art. For example, Teredinibactermay be cultured inaerobic or microanaerobic conditions. Teredinibactermay be grown innatural or synthetic seawater, with a cellulose carbon source, and aninorganic nitrogen source such as ammonium chloride. See, e.g. Waterburyet al., supra; Sipe et al., Bacterial symbiont transmission in the wood-boring shipworm Bankia setacea(Bivalvia: Teredinidae), 66 Applied andEnvironmental Microbiology 1685 (2000).

[0019] Teredinibacter are advantageously grown for the assay on agarplates, but may be grown in any method known in the art including insuspension and in semi-solid suspension.

[0020] The test composition can be administered to the symbiont in anyof a number of ways, including as a liquid extract, as a dry powder, asa solution, as a suspension, and dried on an inert support. A range oftotal amount of test composition is advantageously evaluated. Theconcentration effective for inhibition of the symbiont can be determinedfrom measurements at a plurality of concentrations.

[0021] Any of a number of means known in the art can be used fordetecting inhibition of a symbiont, including Teredinibacter, by a testcompound. Effective methods include detecting a zone of inhibitionsurrounding a discrete site of administration of a composition, whichcan be done on a lawn of bacteria. In other methods the number ofbacterial colonies, or plates, formed in response to the administrationof the composition can be quantitated. Moreover, the rate of growth andthe attenuation or inhibition of growth by administration of the testcomposition can be detected by turbidometric methods. An assay based onmetabolism of substrates can be used to detect changes in growth rate.For example, cellulose derivatized with a marker dye or fluorescent dyecan be used as a substrate. Among these dyes can be ostazin brilliantred h-3b. Release of the dye from insoluble cellulose can indicatemetabolism of the cellulose. The assay can also be based on productionof a metabolic product or inhibition of production of a metabolicproduct. For example, the production of sugars and/or glucose fromdigestion of cellulose can be detected. The invention is not limited tothe exemplary methods provided.

[0022] A composition can be selected that produces a partial inhibitionof the growth or metabolic activity of the symbiont, or total inhibitionof the symbiont. In one embodiment, compositions are effective atinhibiting the symbiont at below ambient temperatures, as may be foundin marine environments. In another embodiment, compositions inhibit thesymbiont at temperatures typically found in estuarine environments. Inyet another embodiment, the compositions inhibit the symbiont attemperatures found on land.

[0023] In one aspect, the invention is a method of wood preservationcomprising treating the wood with at least one of the compositions ofthe invention. The wood can be surface-treated or pressure-treated bymethods standard in the art or by methods readily adaptable by one ofskill in the art. The wood can be treated for about a few minutes, abouta few hours, or about a few days. The composition can be used at anyconcentration, including, but not limited to about 0.01 g/l, about 1.0g/l, and about 100 g/l. In one embodiment, the wood is treated for twodays at two atmospheres with 0.1 g/l of solids in water.

[0024] The invention also comprises a method of wood preservationcomprising treating the wood with the above composition by trapping thecomposition, in whole or in part, in the wood by physical or chemicalmeans. Physical means include treating the wood at elevated temperature,elevated pressure, or both. Chemical means include using co-fixatives,including, but not limited to formaldehyde and carbodiimide, andadjusting the ionic strength, pH, or both. The composition can bebrominated, chlorinated, methylated, or acetylated before or aftertreating the wood. Use of a composition treated to introduce reactivesubstituents is also contemplated. The reactive substituents can be anyknown in the art, including, but not limited to carbene ornitrene-generating substitutents.

[0025] In one aspect, the invention comprises a composition comprisingan extract of a plant, a part of a plant, plant cells, or a wood capableof inhibiting the growth of a cellulolytic symbiont. The symbiont can bea bacterium, including, but not limited to Teredinibacter. In oneparticular embodiment, the composition comprises an extract ofheartwood. In another embodiment, the composition comprises an aqueousextract of heartwood.

[0026] The plant or wood can be any plants or woods havingsymbiont-inhibiting properties, including, but not limited to Douglasfir, ebony, jarrah, koa, lignum vitae, narra, Osage orange, paduccah,purpleheart, red alder, red oak, teak, verawood, western red cedar, orcombinations thereof. Plant cells grown in cell culture are alsosuitable as sources of components of the composition. In one embodiment,cells of symbiont-inhibiting trees are propagated in cell culture. Thecells can be leaf, stem, or root cells. The cells can be propagated byany means known in the art.

[0027] The composition can comprise an aqueous extract. The watercomponent can be tap, distilled, or deionized water. The water can bepotable, but non-potable water is also useful in the invention. Theextractive medium can include salts or minerals and be of any ionicstrength. The salts can be volatile salts such as ammonium bicarbonateor non-volatile salts such as sodium chloride. A variety of pH values ofthe extractive medium are useful in the invention, from about pH 1 toabout pH 14. In one embodiment the pH of the extractive medium isbetween about pH 3 and pH 10. In other embodiments, the pH is betweenabout 3 and pH about 7, or between about 7 and about 10. Any suitableacid or base can be used to adjust the medium. Moreover, the extractivemedium can have water-miscible organic solvents including alcohols orketones, used without or with water.

[0028] In one aspect, the invention is directed to a method foridentifying inhibitors of a cellulolytic organism comprising the stepsof: (a) incubating a cellulolytic organism capable of existing as asymbiont in the absence and presence of a test composition underconditions such that the cellulolytic organism exhibits at least one ofgrowth and metabolic function in the absence of the test composition;and (b) comparing at least one of the growth and the metabolic functionof the cellulolytic organism in the absence and presence of the testcomposition; wherein if at least one of the growth and metabolicfunction in the cellulolytic organism is less in the presence of thetest composition than in the absence of the test composition, the testcomposition comprises an inhibitor of the cellulolytic organism. In themethod, a test composition is incubated with the cellulolytic organism.The incubation may be at any convenient temperature including belowambient temperatures, at about ambient temperatures, or above ambienttemperatures. In one embodiment a temperature of about 4 ° C. to about40° C. is used. In another embodiment a temperature of between about 16°C. and 36° C. is used. In one embodiment a temperature of about 28° C.is used. A suitable temperature is easily selected by one of skill inthe art with a view toward both propagation and detection of inhibitionof an organism capable of existing as a symbiont. Inhibition of eithergrowth or metabolism of the symbiont organism can be determined in anyof a number of ways. The inhibition can be determined by detecting azone of an inhibition, a zone of effect, plaque formation, plaqueinhibition, growth of the organism, inhibition of growth of theorganism, catabolism of substrates, generations of metabolic products,and inhibition of generation of metabolic products. A composition can beselected that provides partial inhibition of the growth or metabolism ofthe organism, or provides total inhibition of the growth or metabolismof the organism. The identified composition is useful as a candidate forthe inhibition of organisms capable of existing as a symbiont. Themethod is suitable for the case in which the organism is capable ofexisting as a shipworm symbiont. Moreover the method is suitable for thesituation where the organism is a Teredinibacter. The method is alsosuitable for the case in which the organism is capable existing as atermite or silverfish symbiont.

[0029] Compositions identified by the above assay are suitable for theinvention. In one embodiment of the invention the compositions do notinclude compositions consisting essentially of a copper-containingcompound or a chromium-containing compound, such as cupric chromate, anarsenic-containing compound such as cupric arsenic, creosote, orcombinations thereof.

[0030] The invention is also directed to a method of wood preservationcomprising treating wood with a composition known to inhibit acellulolytic organism. The treated wood can be used for ship building,for building in an estuarine or marine environment; for building piers,for preparing docks, or any other suitable use. The wood can be treatedby surface treatment such as by application of the composition of theinvention in a paint or spray or stain. The wood to be preserved can inthe alternative be treated under high pressure or elevated temperatureto induce the permeation of the composition of the invention into thefiber of the wood.

[0031] The compositions of the invention include an extract of a plant,a part of a plant, or plant cells, capable of inhibiting the growth of acellulolytic symbiont, and also include any synthetic analogs of theextracts. The compositions of the invention may by synthesized by meansstandard in the art. Moreover, combinations of ingredients are withinthe purview of the invention.

[0032] The invention is also directed to a method for the inhibition ofwood degradation by shipworms comprising contacting wood with aninhibitor of a bacterial symbiont of Bankia setacea, with a proviso thatthe composition does not consist essentially of a copper-containingcompound, a chromium-containing compound, an arsenic-containingcompound, creosote, or combinations thereof. Copper, chromium andarsenic-containing compounds are commonly used in the wood preservingindustry, for example as chromate copper arsenic (CCA), which arethought to be major sources of environmental degradation from theleaching of toxic heavy metals and poisons.

[0033] The use of the singular form in this description is meant toinclude the plural. Thus, for example, “cellulolytic organism” includesa population of the organism, and multiple strains or species, unlessthe text refers to a single organism. By “symbiont” is meant an organismliving, or capable of living in symbiosis with a host organism. Asymbiont is not a fungus as used in this application. The termcellulolytic organism includes organisms that can survive usingcellulose as the primary or only carbon nutrient source.Teredinibacteris an example of a cellulolytic organism. Host organismsshelter cellulolytic organisms in a process that provides metabolicenergy from cellulose for the host.

EXAMPLES Example 1

[0034] Heartwood samples of fourteen wood species (Table 1) are groundto pass a 40 mesh per inch screen. The sawdust samples are oven-dried at104° C. before undergoing the following sequential extraction process.TABLE 1 Selected wood species used in bioassays against Aspergillusniger and a bacterial symbiont of B. setacea. Common Name Latin NameTeak Tectona grandis L. f. Ebony Diospyros ebenum Koenig JarraEucalyptus marginata Sm. Lignumvitae Gualacum sanctum L. PurpleheartPeltogyne spp. Padauk Pterocarpus dalvergioides Roxb. Verawood Bulnesiaarborea (Jacq.) Engler Koa Acacia koa Gray Narra Pterocarpus indiousWilld. Red alder Alnus rubra Bong. Red oak Quercus rubra L. Douglas-firPseudotsuga menziesii (Mirb.) Franco Western redcedar Thuja plicata Donnex. D. Don Osage orange Maclura pomifera (Raf.) C.K. Schneid

[0035] First, a 10 g sub-sample of oven dried sawdust from each woodspecies is placed into a tared beaker along with 100 ml of distilledwater. The beakers are covered with aluminum foil and steamed for 20minutes at 100° C. The slurry is then filtered under suction throughWhatman #4 filter paper and the liquid extract retained for use in thebioassay described below. The residual sawdust is returned to thebeaker, oven-dried for 24 hours at 104° C. and weighed. Next, onehundred ml of hot methanol (60° C.) is added to each of the sawdustsamples. After 60 minutes the resulting extract is collected byfiltration as described above and retained. The sawdust is againreturned to the beaker, dried for 24 hours at 104° C. and weighed.Finally, 100 ml of toluene is added to each of the sawdust samples, thebeakers are covered with Parafilm and the samples are allowed to steepfor 60 minutes. The extract is collected and retained. The sawdust isagain returned to the beaker, dried, and weighed.

[0036] A series of paper discs with increasing levels of extractives areprepared by adding either one, two, three or four 10 μmm l aliquots ofextract to 6 mm diameter BBL Blank Taxo Test Discs (Becton Dickinson andCompany, Cockeysville, Md., 21030, U.S.A. ). The discs are allowed todry between additions. Control discs are treated similarly withdistilled water, methanol and toluene. An additional series of blankdiscs are treated with single 10 μmm l aliquots of 1, 5, 10, 25, 50 and100 ppm solutions of the antibiotic streptomycin sulfate to serve aspositive controls for inhibition of the symbiont.

[0037] Toxicity of the treated paper discs is evaluated in twobioassays. In the first, the discs are placed on the surface of potatodextrose agar (PDA) piates seeded with spores and hyphal fragments ofthe common wood inhabiting mold Aspergillus nigerVan Tiegh according tothe procedures described in the American Society for Testing MaterialsStandard D 5583-94 (ASTM, 1999). This fungus is commonly used fordetecting and quantifying the presence of preservatives in wood and isvery sensitive to low levels of toxicants, particularly phenoliccompounds. In the second bioassay the treated discs are evaluatedagainst the shipworm symbiont seeded on the surface of plates containinga specialized cellulose medium (Table 2). The plates are incubated at28° C. until the fungal spores becomes pigmented or the bacteriumcompletely covers the surface of the plate. At that time, the maximumzone of inhibition (ZOI) and maximum zone of effect (ZOE) are measuredaround each disc to the nearest mm, and the diameter of each disc issubtracted. TABLE 2 Ingredients of a specialized cellulose medium usedfor culturing a bacterial symbiont of B. setacea. Quantity (perIngredient liter) Synthetic sea water^(a) 750.0 ml Distilled water 249.0ml HEPES buffer 5.2 g Sigmacell 101 cellulose 5.0 g Agar 9.0 g K₂HPO₄116.7 mg Na₂CO₃ 99.6 mg Na_MoO₄2H₂O 27.2 mg EDTA disodium salt 4.8 mgFerric ammonium citrate 30.0 mg NH₄Cl 26.8 mg Trace metal mix A-5 +Co^(b) 1.0 ml John Waterbury's VA vitamin mix^(c) 200 μl

[0038]

[0039] A fungus, Aspergillus niger, was used as a control. Water andmethanol extracts have little effect on the growth of Aspergillus niger,while toluene extracts consistently produce a zone of effect that isrelated, in whole or in part, to a residual solvent effect. While thefilter discs are thoroughly aerated prior to fungal exposure, some ofthe solvent remains. Extracts of some wood species produce larger zonesof effect including Douglas-fir, ebony, jarrah, osage orange,purpleheart, red alder, and red oak. A number of these species arehighly durable. Red oak and red alder, however, produce zones of effect.Both of these species latter have little natural resistance to microbialdegradation and are normally rapidly colonized by mold and stain fungi.Thus, these results indicate that the toluene extracts are notappropriate for assessing sensitivity of the test fungus to woodcomponents.

[0040] The results with a bacterial symbiont B. setaceashow inhibitionof symbiont growth. Aqueous extracts from the various woods producemeasurable zones of effect in nearly all instances. Zones of inhibitionranged from 0.2 to 40.3 mm depending upon the extract and wood species.Water extracts applied a single time to the filter paper produce zonesof inhibition (ZOI) greater than 1 mm when extracts are derived fromDouglas-fir, ebony, jarrah, koa, narra, osage orange, paduccah, red oak,and western redcedar. The greatest ZOI's are found with jarrah, koa, redoak, and western redcedar. The ability of red oak to inhibit thesymbiont may be contrasted with the lack of durability exhibited by thisspecies. The effect may reflect tannin extraction, which mightprecipitate bacterial proteins in the membrane, and thereby inhibitmicrobial growth. This latter effect would be expected to be temporaryin nature, because tannins leach relatively rapidly from wood whenimmersed in water. The lack of effect by osage orange at the lowesttreatment level is also interesting since this wood is reported tocontain large quantities of a number of highly toxic extractives.Multiple applications of most of the water extracts of heartwoods,except lignum vitae and verawood, tended to increase the zones ofinhibition for the symbiont. Lignum vitae has a reputation fordurability and the absence of a substantial effect against the symbiontimplies that other mechanisms must account for its durability.

[0041] Zones of inhibition in cultures exposed to methanol extracts ofthe various wood species tend to follow trends that are similar to thosefound with the water extracts. ZOI's greater than 1 mm are found withsingle applications of 12 of the 14 species tested. The largest ZOI'sare found with jarrah, koa, red oak, and western redcedar. The threespecies (jarrah, koa, and western redcedar) exhibit some resistance tomarine borer attack and symbiont inhibition may play a role in thatinhibition. Interestingly, some of these species, notably westernredcedar tend to inhibit shipworm damage well for short periods of time,then fail rapidly, a characteristic suggestive of loss of toxicextractives by leaching over time. Toluene extracts produced noconsistent zones of inhibition for the wood species tested, suggestingthat any effective extracts had already been removed during water andmethanol extraction, or alternatively, that the inhibitors are notsoluble in toluene.

[0042] The symbiont is vertically acquired in the shipworm Bankiasetacea, such that the larvae appear to carry the symbiont to the wood.Sipe et al., supra. The initial settlement of the shipworms on wood,colonization, and growth dependent on digestion of cellulose areindependent processes subject to intervention to disrupt the shipwormcycle.

[0043] Compounds that affect the symbiont may differently affect theability of the marine borer to digest cellulose, to establish newsettlements, and to colonize. Previous reports have established thatshipworms are able to move from an untreated wood member through apreservative treated member and into another. Preventing settlement,halting establishment, and depleting nutrients are each critical aspectsof marine borer control. The tested compositions of wood extracts areclearly effective at inhibiting the symbiont growth and metabolism.Combination of methods to limit settlement or establishment of shipwormswith the methods and compositions of the invention can result in furtherenhancement of protection of wood in marine or estuarine environments.

Example 2

[0044] Acetylation of extracts: An about 50 μmm g portion (dry weight)of an extract having inhibitory properties is stirred in a sealed glassvial with 100 μmm L of acetic anhydride (Supelco) and 100 μmm L of drypyridine for 3 h. The reaction mixture is washed with saturated NaC1,then partitioned between saturated aqueous NaHCO₃ and ethyl acetate. Theorganic, that is, ethyl acetate, layer is collected and concentrated.The product of the acetylation reaction may be significantly less polarthan the starting material by silica gel TLC analysis, indicatingmasking of hydroxyl moieties, carboxyl moieties, or both, by theacetylation reaction.

Example 3

[0045] A solution based assay: A dye-release solution-phase assay isbased on the detection of cellulose-degrading activity based on specificinteraction of direct dyes such as Congo red with polysaccharides. CongoRed reacts with beta- 1,4 -glucans causing a visible red shift (Wood, P.J., Carbohydr. Res., 85:271 (1980) and Wood, P. J., Carbohydr. Res.,94:19 (1981)). A preferred substrate for the test iscarboxymethylcellulose which can be obtained from different sources suchas Hercules Inc., Wilmington, Del., Type 4M6F or Sigma Chemical Company,St. Louis, Mo., Medium Viscosity. The carboxymethylcellulose isincorporated as the main, or sole, carbon source into a minimal agarmedium in quantities of 0.1-1.0%. The effect of the symbiont can bescreened directly on these plates. Symbiont can be pre-treated withputative inhibitor or can be over laid with filter paper impregnatedwith putative inhibitor. Such cellulase-prolucing colonies aredetectable after a suitable incubation time (1-3 days depending on thegrowth), by addition of a 0.1% aqueous solution of Congo Red. Thecoloration is terminated after 20 minutes by removing the dye and adding5M NaCl solution to the plates. The technique can be adapted as acup-plate diffusion assay for the determination ofcarboxymethylcellulase activity in culture filtrates.

Example 4

[0046] The effect of compositions can also be detected in anendoglucanase assay, as follows. The endoglucanase activity isdetermined as the decrease in the viscosity of a solution ofcarboxymethyl cellulose after incubation of symbiont with testcompositions under the following conditions: A substrate solution isprepared, containing 35 g/l carboxymethylcellulose in culture medium.The sample to be analyzed is dissolved or dispersed in the same medium.Then 10 ml substrate solution and 0.5 ml symbiont sample are incubatedat 32° C. for 24 hrs in the absence or presence of 0.5 ml inhibitor.Then the sample is transferred to a viscosimeter (e.g. Haake VT 181, NVsensor, 181 rpm), at a controlled temperature of 32° C. Viscosityreadings are taken at several intervals thereafter. The concentration ofinhibitor product that prevents a decrease in viscosity by one halfunder these conditions is defined as the 50% effective concentration.

[0047] All of the references cited herein are incorporated by referencein their entirety, for all purposes.

[0048] Although the foregoing invention has been described in somedetail for purposes of clarity of understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the invention. It should be noted that there may be alternative waysof implementing both the methods and compositions of the presentinvention. Accordingly, the present embodiments are to be considered asillustrative and not restrictive, and the invention is not to be limitedto the details given herein.

What we claim is:
 1. A method for identifying a composition for limitingor preventing shipworm damage comprising the steps of: (a) incubating ashipworm symbiont in the absence and presence of a test compositionunder conditions such that the shipworm symbiont grows, has metabolicfunction, or both, in the absence of the test composition; and (b)comparing the growth, metabolic function, or both, of the shipwormsymbiont in the absence and presence of the test composition; wherein ifthe growth, metabolic function, or both, in the shipworm symbiont isless in the presence of the test composition than in the absence of thetest composition, the test composition is a composition for limiting orpreventing shipworm damage.
 2. The method of claim 1 wherein thesymbiont is a Teredinibacter.
 3. The method of claim 1, wherein the testcomposition is a wood extract.
 4. The method of claim 3, wherein thewood extract is an aqueous extract from a wood selected from the groupconsisting of Douglas-fir, ebony, jarrah, koa, lignum vitae, narra,Osage orange, paduccah, purpleheart, red alder, red oak, teak, verawood,and western redcedar.
 5. The method of claim 4, wherein the wood isselected from the group consisting of jarra, koa, red oak, and westernredcedar.
 6. A composition identified by the method of claim
 1. 7. Thecomposition of claim 6, wherein the composition does not consistessentially of a copper-containing compound, a chromium-containingcompound, an arsenic-containing compound, creosote, or combinationsthereof.
 8. A method of wood preservation comprising treating the woodwith the composition of claim
 6. 9. A method for identifying acomposition for inhibiting growth, metabolic function, or both, of acellulolytic organism comprising the steps of: (a) incubating acellulolytic organism capable of existing as a symbiont, in the absenceand presence of a test composition under conditions such that thecellulolytic organism grows, has metabolic function, or both, in theabsence of the test composition; and (b) comparing the growth, metabolicfunction, or both, in the cellulolytic organism in the absence andpresence of the test composition; wherein if the growth, metabolicfunction, or both, in the cellulolytic organism is less in the presenceof the test composition than in the absence of the test composition, thetest composition is a composition for inhibiting growth, metabolicfunction, or both, of the cellulolytic organism.
 10. A compositionidentified by the method of claim
 9. 11. The composition of claim 10,wherein the composition does not consist essentially of acopper-containing compound, a chromium-containing compound, anarsenic-containing compound, creosote, or combinations thereof.
 12. Amethod of wood preservation comprising treating the wood with thecomposition of claim
 10. 13. A composition comprising an aqueous extractof a wood wherein the extract is capable of inhibiting the growth,metabolic function, or both, of a cellulolytic organism.